JOURNAL OF PLANT PROTECTION RESEARCH Vol. 56, No. 3 (2016) Chemical constituents and ovicidal effects of mahlab, Prunus mahaleb L. kernels oil on cotton leafworm, Spodoptera littoralis (Boisd.) eggs Hala M. Mead*, Samah N. El-Shafiey, Hend M. Sabry Plant Protection Research Institute, Agricultural Research Center, Dokki, 44516 Giza, Egypt Received: April 1, 2016 Accepted: August 19, 2016 Abstract: The carried out investigations evaluated ovicidal activity of mahlab, Prunus mahaleb L. kernel oil against cotton leafworm, Spodoptera littoralis (Boisd.). The chemical constituents of the fixed oil of mahlab were analyzed using gas-liquid chromatography (GLC). Timnodonic (33.07%), oleic (28.71%) and linoleic (24.35%) were the basic fatty acids, while the major hydrocarbon and sterol were found to be heneicosane (62.57%) and β-sitosterol (10.57%). The LC50 values for the one-day-old egg masses were found to be more susceptible than 3-day-old ones. Moreover, the leaf dip technique occurred to be more efficient than spraying technique. The results also showed abnormalities in the external morphology of egg shell, chorion surface, shell imprints and aeropyles of S. littora- lis eggs treated with mahlab and KZ oils as compared to a control by using scanning electron microscope. Generally, the tested oils significantly reduced the activities of transaminase enzymes (AST and ALT), acid and alkaline phosphatases and total soluble protein except mahlab oil on acid phosphatase as compared to a control. Additionally, the oils of both mahlab and KZ oil affected some bio- logical aspects such as incubation period, larval duration, larval mortality and pupal weight comparing to a control. Key words: electron microscope, enzymes, mahlab, Prunus mahaleb L., Spodoptera littoralis eggs Introduction kernels of P. mahaleb have strong antimicrobial, antifun- The noctuid Spodoptera littoralis (Boisd.) (Lipedoptera: gal and antioxidant activities and may be important for Noctudiae) is a major polyphagous pest, widely distrib- pharmaceutical and industrial purposes. Also, these uted throughout Africa, Mediterranean Europe, and sev- seed kernels are used as sedatives and vasodilators in eral parts of Asia (Hosny and Isshak 1967). Today, Insect Arabic countries (Mariod et al. 2010). In Egypt, the min- Pest Management (IPM) has to deal with the economic eral oils of various qualities are traditionally been used and ecological consequences of the use of pest control for insect pest management as ovicidal agents (Helmy measures. Though highly effective, application of syn- et al. 2012). Therefore, the main goal of this paper was thetic insecticides often has resulted in secondary effects, to utilize the fixed oil of mahlab kernels to control S. lit- which include mammalian toxicity, insect resistance and toralis eggs, whereas KZ oil (mineral oil) was used as ecological hazards. The diversification of the approach a reference. The chemical composition of mahlab oil inherent in IPM is necessary for better environmental components were also analyzed and identified. Fur- protection. Among the alternative strategies, the use of thermore, the ovicidal activities, changes in the mor- plants insecticidal allelochemicals appears to be promis- phological structure of eggs; biochemical and biological ing, and many of these compounds are secondary plant features induced by mahlab and KZ oils on S. littoralis substances, including alkaloids; quinones and oils (Reg- were studied. nault-Roger 1997). Oils from plants may have minimal direct and/or indirect effects on natural enemies for ecological equi- Materials and Methods librium. For these reasons, oils are currently under in- Tested oils vestigation for their broad-spectrum pest control prop- erties. Mahlab, Prunus mahaleb L. (Rosales: Rosaceae) Prunus mahaleb oil is a large perennial shrub or deciduous small tree that is found through Mediterranean countries. Due to the A sample of about 200 g of kernels of mahlab, P. maha- special fragrance, previous studies focused on the seed leb was bought at a local market, Sharqia Governorate, kernels, which have a high protein content and fixed oil Egypt. (Mariod et al. 2009). Özçelik et al. (2012) found that seed *Corresponding address: [email protected] 280 Journal of Plant Protection Research 56 (3), 2016 Extraction technique of fixed oil 3-day-old eggs. Two different techniques were used; leaf dip technique (dipped for 20 s) and spraying technique. Extraction of P. mahaleb oil was performed at room tem- Five egg masses were used for each tested concentration perature using electric grinder. About 200 g of ground of mahlab and KZ oil in both tested groups. The treated kernels were soaked in 300 ml petroleum ether (60–80°C) egg-masses were left to dry in air, and then transferred for 3 days. to Petri dishes (5 egg-masses/dish). The same number of The pooled extract was filtered and evaporated un- egg-masses was dipped and sprayed with distilled water der vacuum at 50°C to yield about 80 ml oil residue, that as a control. Daily inspection for all treatments was per- stored at 4°C until use. formed until the untreated eggs hatched. Two controls were used in this experiment. Petroleum Determination of fatty acids ether alone (solvent) was the positive (+ve) control to en- sure that the effect against egg-masses attribute to only Free fatty acids were separated from crude oil as methyl- the active compounds in the mahlab oil, while distilled ated at the Food Technology Research Institute, Center water was used as a negative (-ve) control for KZ oil. The Laboratory, Egypt according to the method by Cunniff mortality percentages of egg-masses were recorded as av- (1995) and determined using gas liquid chromatography erage mortality percentages of each tested concentration analysis (Macherey-Nagel, Düren, Germany) according using Abbott’s formula (1925). To estimate the LC50 val- to the procedure reported by Arens et al. (1994). ues, the corrected mortality percentages were subjected to probit analysis according to Finney (1952). Isolation of unsaponifiable fractions Concerning the susceptibility of the two ages of egg-masses, the LC50 values of mahlab treatments and About 10 ml of crude oil was refluxed with 100 ml of 10% KZ oils against 3-day-old egg-masses of S. littoralis us- alcoholic potassium hydroxide for 6 h, and the obtained ing leaf dip technique were evaluated. As for scanning residue was diluted with ether (5 × 300 ml). The combined electron microscopy and biochemical analysis, the data ether extract was washed several times with water to re- was recorded 48 h post treatment with the previous de- move alkalinity, dried over anhydrous sodium sulphate scribed LC50s, to ensure the formation of embryos inside and let to evaporate. The sample of 2 g of unsaponifiable treated eggs. matter was collected. Analysis of unsaponifiable com- pounds was carried out in gas-liquid chromatography Scanning electron microscopy examination (GLC) at the National Research Center, Cairo, Egypt ac- cording to the method described by Ramadan and Mörsel Scanning electron microscopy of egg shell (chorion) of (2003). S. littoralis using the most potent LC50s of both mahlab and KZ oil and petroleum ether treatment were used. KZ mineral oil® (95% EC) Preparation of tissue samples for scanning A petroleum-derived oil, produced by Kafr El-Zayat Co. electron microscopy examination for Pesticides & Chemicals, Egypt was used in the study at the rate of 15 ml · l–1 water. The tested egg-masses were fixated by glutaraldehyde 2.5% and dehydrated by serial dilutions of ethanol us- Culture of the cotton leafworm, Spodoptera littoralis ing automatic tissue processor (Leica EM TP), then the samples were dried using CO rearing technique 2 critical point drier (Tousi- mis Audosamdri-815). Next the samples were coated by A laboratory (susceptible) strain of S. littoralis was reared gold coater (SPI-Module) and examined by scanning elec- away from any insecticidal contamination, at the Plant tron microscopy (JEOL-JSM-5500 LV) using high vacuum Protection Research Institute Zagazig, Sharqia Governor- mode at Regional Center of Mycology and Biotechnology, ate, Egypt. Egg-masses were reared on leaves of castor Cairo, Egypt. bean, Ricinus communis L. according to El-Defrawi et al. (1964) at 26±1°C and 70±5% relative humidity (RH). Biochemical studies Susceptibility of two developmental egg stages of Samples preparation Spodoptera littoralis Egg-masses used for biochemical assays were collected Two different techniques were used to study the suscepti- 48 h after treatment with potent LC50s of tested oils plus bility of eggs at the two developmental ages (1- and 3-day- positive control. Five mg of egg-masses per treatment old). Six concentrations of mahlab oil were prepared using were homogenized in distilled water using a Teflon ho- petroleum ether as a solvent (10.0, 5.0, 2.5, 1.25, 0.625, and mogenizer surrounded with a jacket of crushed ice for 0.312%) (v/v). As for KZ oil, six serial concentrations start- 3 min. Homogenates were centrifuged at 3,500 rpm for ing with the recommended one (1.5 l · 100 l–1 water) were 10 min at 5°C to remove haemocytes. The supernatants used. Egg-masses were collected from laboratory reared were used directly for the biochemical analysis. Three population and divided into two groups; the first one replicates were used for each assay. was the 1-day-old eggs and the second group contained Chemical constituents and ovicidal effects of mahlab, Prunus mahaleb L. kernels oil on cotton leafworm… 281 Total soluble protein (p < 0.05) (Snedecor and Cochran 1980). Data were sub- jected to statistical analyses using the software package Colorimetric determination of total soluble protein in the Costat® Statistical Software (2005) a product of Cohort total homogenate of larvae was carried out as described Software, Monterey, California, USA. by Gornall et al. (1949). Results Transaminase enzymes Fatty acid composition Aspartate aminotransferase (AST) and alanine amino- transferase (ALT) enzyme activities were determined The fatty acid composition of P.